Dark or heterotrophic fermentation by anaerobes as well as some microalgae, such as green algae on carbohydrate-rich substrates, can produce hydrogen in an anaerobic environment without the need of light energy (Zhang et al., 2006; 2007a, b, c; 2008a, b, c, d; Show et al., 2007; 2010; Lee et al., 2011). The possibility with dark fermentative hydrogen production from algal biomass remains that hydrogen was produced by heterotrophic bacterial satellites present in the algal biomass slurries (Carver et al., 2011; Lakaniemi et al., 2011). It has been well established that methane is generated in conventional anaerobic fermentation in two distinct stages: acidification and methane production. Each stage is carried out by specific microorganisms through syntrophic interactions. Hydrogen is produced in the first-stage

acidogenesis as an intermediate metabolite, which in turn is used as an electron donor by methanogens at the second-stage methanogenesis.

Formation of molecular hydrogen in dark fermentation is generally accom­plished through two pathways in the presence of specific coenzymes (Show et al.,

2011) . One pathway is by a formic acid decomposition route; the other pathway is the reoxidization of nicotinamide adenine dinucleotide (NADH) route represented by NADH + H+ + 2Fd2+! 2H+ + NAD+ + 2Fd+ and 2Fd2+ + 2H+! 2Fd+ + H2 under the mediation of hydrogenase. The Embden-Meyerhof, or glycolytic, pathway is un­doubtedly the most common route for glucose degradation to pyruvate, which functions in the presence or absence of oxygen (Prescott et al., 2002). In this pathway, glucose is converted into pyruvate associated with the conversion of NADH from NAD+ via an­aerobic glycolysis represented by C6H12O6 + 2NAD+! 2CH3COCOOH + 2NADH + 2H+. Electron transfer via pyruvate-ferredoxin oxidoreductase or NADH-ferredoxin oxidore- ductase and hydrogenase could be affected by the corresponding NADH and acetyl-CoA levels or prevailing environmental conditions. Thus, the oxidation-reduction state has to be balanced through the NADH utilization to form several reduced compounds, i. e., lactate, ethanol, and butanol, resulting in a lowered hydrogen yield.

Theoretically, it is possible to harvest hydrogen at the acidogenesis stage of anaerobic fer­mentation if only acidogens are left to produce hydrogen gas and other metabolites and the final methanogenesis stage and other hydrogen-consuming biochemical reactions are inhibited during the dark fermentation. However, inhibition of hydrogen-consuming micro­organisms in complex microbial consortia decomposing algal biomass for hydrogen produc­tion poses a challenging task. It has been reported that the hydrogen produced from green algae C. vulgaris and D. tertiolecta biomass by anaerobic enriched cultures containing BESA was subsequently consumed by nonmethanogenic microorganisms (Lakaniemi et al.,

2011) . Similar hydrogen utilization was also reported from the work on hydrogen production by anaerobic sludge fed with lipid-extracted Scenedesmus algal biomass (Yang et al., 2010).

During the dark fermentation, carbohydrates are converted into hydrogen gas and volatile fatty acids and alcohols, which are organic pollutants and energy carriers. For the purpose of energy production and protection of the water bodies, a second-stage process is necessary to recover the energy residues remaining in the effluent in the form of fatty acids and alcohols. Thus the fermentative reactor becomes part of a process wherein the effluent post-treatment process and hydrogen utilization should also be included. A possible second-stage process is photofermentation, anaerobic digestion, or microbial fuel cells, which have been assessed in a recent review (Show et al., 2012).

For hydrogen produced from dark fermentation to be used alone in an internal combustion engine or a fuel cell, some issues such as biohydrogen purification, storage, and transport are to be addressed. Unlike a biophotolysis process that produces only hydrogen, the gaseous product of dark fermentation is a mixture of primary hydrogen (generally less than 70%) and CO2 but may also contain other gases such as CH4, H2S, ammonia, and/or moisture. Pu­rification of the hydrogen is essential before the hydrogen utilization can be practical (Show et al., 2011; 2012). Nevertheless, hydrogen production by dark fermentation is an attractive process in the sense that it does not demand large land space and is not affected by weather conditions (solar radiation is not a requirement). Also, among the biohydrogen production processes, dark fermentation is deemed to be more favorable. Hydrogen is yielded at a high rate, and various organic compounds and wastewaters are enriched with carbohydrates as the substrate results in low-cost hydrogen production (Hallenbeck and Ghosh, 2009). Hence, the feasibility of the technology yields a growing commercial value.